The human genome has been sequenced for a decade, but solving how proteins fold and assemble into complexes remains a challenge. More than half of all proteins -- including 95% of integral membrane proteins -- do not crystallize and thus their structures cannot be determined by crystallography. Our project addresses this problem by creating an instrument that can determine atomic-resolution structures of individual biological macromolecules without requiring crystallization. We propose to merge four distinct technologies that should allow structures of macromolecules up to a megaDalton to be resolved at high resolution (better than 2 ) in a few hours. The key steps are a) to electrospray and purify macromolecules by mass spectrometry, b) to quickly chill these macromolecules to near absolute zero temperature with superfluidic helium droplets, c) to controllably orient several thousand chilled macromolecules to within ~1 for 50 s using intense elliptically polarized IR laser light while confining them in a small diffraction zone, and d) to collect continuous diffraction images from these oriented macromolecules using a pulsed electron beam. Steps c) and d) will be repeated for each orientation to span the reciprocal space at 1 intervals by rotating the polarization of the laser. The continuous diffraction images provide sufficient information to directly calculate phases by well-established oversampling methods thereby directly yielding electron density maps. In this grant period, our goal is to demonstrate the proof-of-concept by recording anisotropic electron diffraction images from laser aligned protein ions embedded in superfluid helium droplets. Further development will address the resolution and quality of data issues with major improvements in experimental hardware. This idea is based on recent breakthroughs in several disciplines. A large body of evidence has established that protein complexes can retain their conformation, remain associated in large multimeric complexes and keep ligands bound in vacuo after electrospray ionization. Capitalizing on recent advances in laser-induced alignment at superfluid helium temperatures (0.37 Kelvin), our proposed instrument will instantaneously freeze macromolecules, allowing them to be oriented within 1 in all three Euler angles by a 200,000 V/cm electric field generated by the IR laser. Ultimately, this approach will allow structures to be determined at high resolution in a few hours from a few nanomoles of partially purified complexes of proteins that are otherwise inaccessible by current methods. If successful, this instrument will reshape the landscape of structural biology, transform structure-based drug screening, allow rapid determination of the effects of mutations on structure, and open new realms of biophysics to understand the effects of solvent on structure.